p-tert-butylstyrene

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 2018 - 06 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Synonyms: p-tert-butylstyrene
CAS No: 1746-23-2
Purity: 96.4%

Analytical monitoring:

yes

Details on sampling:

- Nominal concentrations: 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L
- Mean measured concentrations: 0.030, 0.069, 0.14, 0.27, and 0.57 mg/L
During the in-life phase of the definitive study, water samples were removed from a single replicate of each treatment and the control, with the sampled replicate alternating at each successive sampling (replicate A, then B, then C, etc.). Samples were removed on test day 0, 4 (hatch), 11, 18, 25, and 32. Additional subsamples of the test solutions were also collected at each sampling interval and stored frozen as archive samples. All exposure samples were removed from the approximate midpoint of each aquarium using a pipet. In addition, a sample of the stock solution was analyzed at each sampling interval during the exposure period.
The exposure samples were analyzed for p-tert-butylstyrene using methodology validated at Smithers Viscient.

Vehicle:

yes

Remarks:

Dimethylformamide (DMF, CAS No. 68-12-2)

Details on test solutions:

A 200 mg/mL diluter stock solution was prepared prior to exposure initiation and as needed thereafter throughout the definitive exposure by adding, for example, 10.3880 g of p tert butylstyrene (10.0140 g as active ingredient) to a 50-mL volumetric flask and then bringing it to total volume with dimethylformamide (DMF, CAS No. 68-12-2). The resulting stock solutions were observed to be clear and colorless following mixing by inversions.

A 38 µL/mL solvent stock solution was prepared prior to exposure initiation and as needed throughout the definitive exposure thereafter by diluting 38 mL of DMF to 1000 mL with deionized water. The resultant solvent stock solutions were observed to be clear and colorless following preparation.

Test organisms (species):

Pimephales promelas

Details on test organisms:

Fathead minnow embryos (SMV Lot No. 18A345) used during this testing were obtained from brood stocks maintained at Smithers Viscient. The brood stocks used were between four and seven months old. No mortality was observed among the brood stock during the seven days prior to testing. The water flowing to the culture unit was from the same source as the dilution water used during the early life-stage exposure. During the 14 days prior to test initiation, the culture temperature ranged from 23 to 25 °C and the dissolved oxygen ranged from 90.1 to 102% of saturation.

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

32 d

Hardness:

64-86 mg/L CaCO3

Test temperature:

24-26°C

pH:

7.1-7.2

Conductivity:

500-680 µS/cm

Nominal and measured concentrations:

- Nominal concentrations: 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L
- Mean measured concentrations: 0.030, 0.069, 0.14, 0.27, and 0.57 mg/L

Details on test conditions:

Prior to exposure initiation, a Harvard Apparatus pump, in conjunction with a 10.0-mL Hamilton gas-tight syringe, was calibrated to deliver 0.00969 mL/cycle of the 200 mg/mL diluter stock solution into the diluter system’s chemical mixing chamber, which also received 1.938 L of dilution water per cycle. The mixing chamber was positioned over a water-driven magnetic stir plate and was partially submerged within an ultrasonic water bath. The continuous stirring (with a Teflon coated stir bar) and sonication aided the solubilization of the test substance with the dilution water. The solution in the mixing chamber was equivalent to that of the highest nominal test concentration (1.0 mg/L) and was subsequently diluted by a constant factor of 2.0 to produce the remaining nominal test concentrations (0.50, 0.25, 0.13, and 0.063 mg/L).

The concentration of DMF in the solution in the mixing chamber, which also served as the high test concentration, constituted the highest DMF concentration (5.0 µL/L). A Fluid Metering (FMI) pump was calibrated to deliver 0.674 mL/cycle of the 38 µL/mL solvent stock solution to 5.085 L of dilution water per cycle which was subsequently delivered to the solvent control and treatment vessels. The DMF concentration in the solvent control and the treatment levels was 5.0 µL/L, which was equal to that in the high test concentration.

A set of control aquaria was also established which contained the same dilution water and was maintained under the same conditions as the treatment aquaria, but contained no p tert butylstyrene or DMF.

The exposure was conducted using a system consisting of an intermittent-flow proportional diluter (Mount and Brungs, 1967), a temperature-controlled water bath, and a set of 28 exposure aquaria. The exposure system was designed to provide five concentrations of the test substance, a control, and a solvent control to four replicate exposure aquaria. Flow-splitting cells were employed to equally distribute the solutions to the four replicate aquaria at a rate of approximately 250 mL of test solution per aquaria per cycle (see Appendix 3 for further details). Flow splitting accuracy of the diluter cells was within 10% of the nominal value.

The diluter system was calibrated prior to exposure initiation and calibration was confirmed at exposure termination by measuring delivery volumes of toxicant and dilution water. The function of the diluter system (e.g., flow rate, stock solution consumption) was monitored twice daily and a visual check of the system's operation was performed twice daily. In addition, analysis of the exposure solutions for p-tert-butylstyrene concentration was also used to verify proper operation of the diluter system. The exposure system was functioning properly for 26 days prior to exposure initiation. This extended equilibration time was designed to allow equilibration of the test substance in the diluter apparatus and exposure aquaria and to monitor the behavior of the test substance over time (see Appendix 3 for further details). The exposure aquaria were labeled to identify the nominal test substance concentration/control designation and designated replicate (A, B, C, and D).

The diluter system and exposure aquaria were fabricated of glass and silicone sealant. Each 2.5 gallon exposure aquaria measured 30 × 14.5 × 20 cm with a 12.5-cm high side drain that maintained a constant exposure solution volume of approximately 5.5 L. During the 32-day exposure, the aquaria were brushed and siphoned weekly to remove excess food and fecal matter.
Embryo incubation cups were round glass jars (5 cm) diameter, 8 cm high with 475-micron mesh opening Nitex screen bottoms. A rocker arm apparatus, driven by a 1-rpm electric motor, as described by Mount (1968), was used to gently oscillate the incubation cups in the test solutions. The diluter system delivered the control and exposure solutions to the exposure aquaria at a rate sufficient to provide approximately 10 aquarium volumes per 24-hour period, with a 90% replacement time of approximately five hours (Sprague, 1969). Due to the reported volatility of the test substance, this maximum turnover rate was utilized in order to mitigate the loss of test concentrations.
The aquaria were impartially positioned in a water bath containing circulating water designed to maintain the test solution temperatures at 25 ± 1.5 °C. Two Process Technology USA immersion heaters regulated by an Omega CN76000 temperature controller were used to maintain temperatures. Illumination was provided by Sylvania Octron fluorescent lights centrally located above the test aquaria. A 16-hour light photoperiod at a light intensity range of 57 to 120 footcandles (620 to 1300 lux) and 8 hours darkness with a 15- to 30 minute transition period was provided at the exposure solution’s surface. Light intensity was measured with a Fisher Scientific Traceable light meter.

Reference substance (positive control):

no

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.03 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.069 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

length

Details on results:

Based on mean measured concentrations and total length, which was the most sensitive endpoint, the NOEC was determined to be 0.030 mg/L and the LOEC was determined to be 0.069 mg/L.

For this study, ECx values were calculated for total length and wet weight. For the remaining endpoints (embryo hatching success; percent of live, normal larvae at completion of hatch and larval survival), a significant lack of fit or failing residual normality and variance was observed in all relevant statistical models. Therefore, ECx values were not used to quantify treatment effects in these endpoints.

Reported statistics and error estimates:

Data on embryo hatching success, percentage of embryos producing live, normal larvae at hatch, larval survival, and larval growth at exposure termination (total length and wet weight) were statistically analyzed to establish treatment level effects. Statistical analyses followed the procedures described in 'Current Approaches in the Statistical Analysis of Ecotoxicity Data (OECD, 2006)' and OECD Guideline 210.

Analyses were performed using mean organism response in each replicate aquarium. All statistical analyses were conducted at the 95% level of certainty except in the case of the qualifying tests for normality and homogeneity of variance, in which the 99% level was applied:

1. Solvent control and control data were compared using an Equal Variance Two-Sample t-Test. If no significant difference was established, the pooled control was used for comparison of the treatment responses.

2. Statistical analysis of percent embryo hatching success, percent live normal larvae at hatch and percent larval survival was performed. Since the data for these endpoints was non-monotonic, Fisher’s Exact Test with Bonferroni Holm’s Adjustment was used to determine treatment effects.

3. Shapiro Wilks’ Test for normality was conducted to evaluate the distribution of the data.

4. Bartlett’s Equality of Variance Test was conducted to evaluate the homogeneity of the data.

5. Since total length and wet weight data met the assumptions for both normal distribution and homogeneity of variance and were monotonic, Williams’ Multiple Comparison Test was used to establish treatment effects for these endpoints.

CETIS Version 1.8 was used to perform the statistical computations. The lowest mean measured concentration that elicited a statistically significant reduction on organism performance (LOEC) and the highest mean measured concentration that did not elicit a statistically significant reduction between the exposed organisms and the control (NOEC) were determined.

Validity criteria fulfilled:

yes

Conclusions:

Based on mean measured concentrations and total length, which was the most sensitive endpoint, the NOEC was determined to be 0.030 mg/L and the LOEC was 0.069 mg/L (mean measured).

Executive summary:

A study was conducted to evaluate the effects of the test substance on embryos and larvae of a sentinel freshwater fish species, the fathead minnow (Pimephales promelas), under flow-through conditions according to OECD Guideline 210, in compliance with GLP. Approximately 18 h old fish embryos were exposed at nominal concentrations of 0, 0.063, 0.13, 0.25, 0.50 or 1.0 mg/L of the test substance. Following initiation of exposure, dead and live embryos were counted daily until the day of hatch (Day 3). Day 4 was considered to be the day of completion of hatch. Calculations of hatching success were based on the number of live, dead or deformed larvae per incubation cup after hatching was complete compared to the number of embryos per cup on Day 0. The 28 d post-hatch larval exposure was initiated following completion of hatching. On Day 4, the surviving larvae present in each incubation cup were sorted into 20 organisms per replicate/80 organisms per treatment level or control and placed into each respective exposure aquarium. During the post-hatch exposure period, dead larvae were removed when observed, and behaviour and appearance of the larval fish were observed and recorded daily. Larval survival was estimated daily. Beginning on Day 5 (Day 1 post-hatch), the larvae were fed live brine shrimp nauplii (Artemia salina) three times daily. At each feeding, larvae were fed ad libitum such that all larvae were afforded equal access to food. Larvae were not fed during the 24 h period prior to study termination. At 28 d post-hatch, the exposure was terminated. The surviving larvae in each replicate aquarium were euthanized, counted to determine larval survival and measured individually to determine total length and wet weight. The larvae were measured and weighed. The mean and standard deviation were calculated using individual measurements (total length and wet weight) for each replicate aquarium. At termination, individual wet weights were also determined for the control larvae to determine biomass loading. Analytical dose verification was conducted using gas chromatography with mass selective detection (GC‑MSD). Mean measured concentrations corresponded to 0, 0.030, 0.069, 0.14, 0.27 and 0.57 mg/L. Based on mean measured concentrations and total length (considered as the most sensitive endpoint), the NOEC was determined to be 0.030 mg/L and the LOEC was 0.069 mg/L. EC_xvalues were calculated for total length and wet weight. For the remaining endpoints (embryo hatching success; percent of live, normal larvae at completion of hatch; and larval survival), a significant lack of fit or failing residual normality and variance was observed in all relevant statistical models. Therefore, EC_xvalues were not used to quantify treatment-related effects for these endpoints (Urann, 2018).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.03 mg/L

Additional information

A study was conducted to evaluate the effects of the test substance on embryos and larvae of a sentinel freshwater fish species, the fathead minnow (Pimephales promelas), under flow-through conditions according to OECD Guideline 210, in compliance with GLP. Approximately 18 h old fish embryos were exposed at nominal concentrations of 0, 0.063, 0.13, 0.25, 0.50 or 1.0 mg/L of the test substance. Following initiation of exposure, dead and live embryos were counted daily until the day of hatch (Day 3). Day 4 was considered to be the day of completion of hatch. Calculations of hatching success were based on the number of live, dead or deformed larvae per incubation cup after hatching was complete compared to the number of embryos per cup on Day 0. The 28 d post-hatch larval exposure was initiated following completion of hatching. On Day 4, the surviving larvae present in each incubation cup were sorted into 20 organisms per replicate/80 organisms per treatment level or control and placed into each respective exposure aquarium. During the post-hatch exposure period, dead larvae were removed when observed, and behaviour and appearance of the larval fish were observed and recorded daily. Larval survival was estimated daily. Beginning on Day 5 (Day 1 post-hatch), the larvae were fed live brine shrimp nauplii (Artemia salina) three times daily. At each feeding, larvae were fedad libitumsuch that all larvae were afforded equal access to food. Larvae were not fed during the 24 h period prior to study termination. At 28 d post-hatch, the exposure was terminated. The surviving larvae in each replicate aquarium were euthanized, counted to determine larval survival and measured individually to determine total length and wet weight. The larvae were measured and weighed. The mean and standard deviation were calculated using individual measurements (total length and wet weight) for each replicate aquarium. At termination, individual wet weights were also determined for the control larvae to determine biomass loading. Analytical dose verification was conducted using gas chromatography with mass selective detection (GC‑MSD). Mean measured concentrations corresponded to 0, 0.030, 0.069, 0.14, 0.27 and 0.57 mg/L. Based on mean measured concentrations and total length (considered as the most sensitive endpoint), the NOEC was determined to be 0.030 mg/L and the LOEC was 0.069 mg/L. EC_xvalues were calculated for total length and wet weight. For the remaining endpoints (embryo hatching success; percent of live, normal larvae at completion of hatch; and larval survival), a significant lack of fit or failing residual normality and variance was observed in all relevant statistical models. Therefore, EC_xvalues were not used to quantify treatment-related effects for these endpoints (Urann, 2018).